Cancer and Metastasis
Cancer is a growing problem in the world, particularly in the western countries. The increase in cancer cases and in cancer-related mortality may be attributed, at least in part, to an overall decrease in the rate of deaths from other causes, such as infectious disease. Therefore, new treatments for cancer are becoming increasingly important, both in order to extend the lifespan and also to increase quality of life.
The mechanism basis of the ability of metastatic cells to home and proliferate in the parenchyma of certain organs, such as the liver, and to develop organ-specific metastases remain largely unknown. For metastasis to occur, the malignant cells must escape from the primary tumor, circulate through the blood stream and subsequently arrest and develop in the target tissues. Recently, it was shown that metastatic breast carcinomas utilize the SDF-1/CXCR4 chemokine/chemokine receptor pathway for metastasis (1-3).
In recent years, chemokines, molecules that actively modulate the onset and progression of the immune response, and their cellular receptors have received increasing attention due to their critical role in the progression of immune disease states such as Asthma, Atherosclerosis, Graft Rejection, AIDS, and Multiple Sclerosis (MS). Chemokines are a family of structurally related proteins that have an essential role in the recruitment and activation of cells from the immune system. Thus, chemokines can be considered as master regulators of the body's immune response repertoire. Because of their varied activities, chemokines are potentially valuable targets for therapeutic intervention in a wide range of diseases (4).
Several research groups have shown anti-tumor activity with a variety of chemokines overexpressed in tumor cells. More specifically, anti tumor activity was shown for MCP-3, MIP-1alpha, Rantes, lymphotactin, TCA-3, and MIP-3alpha (5). The chemokine receptor CXCR-4 has been shown to function as the major co-receptor for HIV-1/2 on T cells, as well as the CD4-independent receptor for HIV-2 (6). The murine CXCR-4-predicted amino acid sequence is 91% identical to human CXCR-4. CXCR-4 is expressed on human CD34+ stem cells, PBLs, monocytes, and neutrophils (7). Stromal cell-derived factor 1 alpha/beta (SDF-1), the ligand for CXCR-4, is a powerful chemoattractant for T cells and CD34+ cells and can inhibit HIV infection of these cells (8). Human and murine SDF-1 differ by one amino acid and are cross-reactive. SDF-1 is produced in high levels in the bone marrow, lymph node (LN) and spleen (9-11). In contrast to pro-inflammatory chemokines, SDF-1 expression is not regulated by stimuli generated by viral or bacterial infections, suggesting a major role for SDF-1 in steady-state homeostatic processes, such as leukocyte trafficking (12).
SDF-1 can induce the arrest of rolling CD34+ on human endothelium under shear flow in vitro, and that in vivo, human bone marrow endothelial cells express SDF-1 (13). Furthermore, by increasing the expression level of CXCR4 on CD34+ progenitors, their ability to migrate to and engraft in the bone marrow is improved (14). Overexpression of human CXCR4 on murine T cells led to enhanced numbers of these cells in the murine BM and to a dramatic decrease in their numbers in the circulation (15). In addition, injection of SDF-1 into the murine spleen and bone marrow was shown to increase the homing of FDCP-mix cells to the spleen and the homing of human CD34+ cells to the bone marrow (16). These results suggest that an increase in the concentration of SDF-1 within the bone marrow microenvironment or enhanced expression of CXCR4 on effector T cells may stimulate the homing and retention of these cells to the bone marrow.
The process of metastasis requires at least three consecutive steps in which chemokines may be involved. First, chemokines may facilitate the interaction of tumor cells with endothelial cells. Second, following the transendothelial migration of tumor cells chemokines can direct the intra-tissue localization of tumors. Thereafter chemokines may stimulate the growth of tumor cells after metastasis.
Saccharide-based Compounds
Heparin is a glycosaminoglycan, a polyanionic sulfated polysaccharide, which is used clinically to prevent blood clotting as an antithrombotic agent. In animal models, heparin has been shown to reduce the ability of autoimmune T lymphocytes to reach their target organ (Lider, O. et al., Eur. J. Immunol. (1990) 20:493-499). Heparin was also shown to suppress experimental autoimmune diseases in rats and to prolong the allograft survival in a model of skin transplantation in mice, when used in low doses (5.mu.g for mice and 20.mu.g for rats) injected once a day (Lider, O. et al., J. Clin. Invest. (1989) 83:752-756).
The mechanisms behind the observed effects are thought to involve inhibition-of release by T lymphocytes of enzyme(s) necessary for penetration of the vessel wall, primarily the enzyme heparanase that specifically attacks the glycosaminoglycan moiety of the sub-endothelial extracellular matrix (ECM) that lines blood vessels (Naparstek, Y. et al., Nature (1984) 310:241-243). Expression of the heparanase enzyme is associated with the ability of autoimmune T lymphocytes to penetrate blood vessel walls and to attack the brain in the model disease experimental autoimmune encephalomyelitis (EAE).
European Patent Application EP 0114589 (Folkman et al.) describes a composition for inhibition of angiogenesis in mammals in which the active agents consist essentially of (1) heparin or a heparin fragment which is a hexasaccharide or larger and (2) cortisone or hydrocortisone or the 11-.alpha. isomer of hydrocortisone. According to the disclosure, heparin by itself or cortisone by itself are ineffective; only the combination of both gives the desired effects. Although there is no proof in the literature that there is a connection between angiogenesis and autoimmune diseases, the description on page 5 of the patent application connects angiogenesis with psoriasis and with arthritis, indicating the use of high doses of 25,000 units to 47,000 units of heparin per day (i.e., about 160 to about 310 mg per day).
Horvath, J. E. et al., in Aust. N.Z.J. Med. (1975) 5(6):537-539, describe the effect of subanticoagulant doses of subcutaneous heparin on early renal allograft function. The daily dosage is high (5000 U or about 33 mg) and the conclusion of the study is that heparin in subanticoagulant doses has no effect on early graft function or graft survival and that it may be associated with increased hemorrhagic complications.
Toivanen, M. L. et al., Meth. and Find. Exp. Clint. Pharmacol. (1982) 4(6):359-363, examined the effect of heparin in high dosage (1000 U/rat or about 7 mg/rat) in the inhibition of adjuvant arthritis in rats and found that heparin enhanced the severity of the rat adjuvant arthritis.
PCT Patent: Application PCT/AU88/00017 published under No. WO88/05301 (Parish et al.) describes sulphated polysaccharides that block or inhibit endoglycosylase activity, such as heparanase activity, for use as antimetastatic and anti-inflammatory agents. Heparin and heparin derivatives, such as periodate oxidized, reduced heparins, that had negligible anticoagulant activity, were shown to have antimetastatic and anti-inflammatory activity when used in dosages within, the range of 1.6-6.6 mg per rat daily, administered by constant infusion (corresponding to 75-308 mg daily for an adult human patient).
Heparin and heparan sulfate are closely related glycosaminoglycan macromolecules. The degradation products of these polymeric macromolecules, which are termed low molecular weight heparins (LMWH), may have the same or greater pharmacological effects on the blood clotting system as the parent macromolecules. Furthermore, because there is extensive but incomplete post-synthetic processing of the polymer's basic disaccharide subunit, glucuronic acid and N-acetyl glucosamine, the LMWH will be a heterogeneous mixture not only of sizes but also of chemical compositions (See Goodman and Gilman's The Pharmacological Basis of Therapeutics, 8th Ed., (Pergamon Press, New York, 1990) pp. 1313-1315. Methods to obtain low molecular weight products from heparin, which are useful as anticoagulants, are described in the art. These methods seek to optimize the persistence in vivo or the extent of hemorrhagic side effects of their products (See, for example, Alpinro R. R., et al., U.S. Pat. No. 5,010,063; Choay, J., et al., U.S. Pat. No. 4,990,502; Lopez, L. L., et al., U.S. Pat. No. 4,981,955). Others teach the use of affinity chromatographic methods to obtain low molecular weight products (See, for example, Rosenberg, R. D., et al., U.S. Pat. No. 4,539,398 and Jordan, R. E., et al., U.S. Pat. No. 4,446,314).
Psuja, P., as reported in Folio Haematol. (Leipz), (1987) 114:429-436, studied the effect of the heterogeneity of heparins on their interactions with cell surfaces. Psuja reported that there are moderate affinity receptors for LMWH found on cultured endothelial cells, but he-determined that the upper limit of the fraction of LMWH bound to these receptors was less than 1% of total LMWH.
Other workers have demonstrated effects of LMWH on the metabolism of a variety of cultured cell types. Asselot-Chapel, C., et al., in Biochem. Pharmacol. (1989) 38:895-899 and Biochem. Biophys. Acta, (1989) 993:240-244, report that LMWH cause cultured smooth muscle cells to decrease the ratio of type III to type I collagen and fibronectin synthesis. Rappaport, R. in U.S. Pat. No. 4,889,808, teaches that LMWH can cause human diploid pulmonary fibroblasts, cultured in the absence of serum, to respond to LMWH by increased secretion of tissue plasminogen activator and related proteins.
Effects of LMWH on complex multicellular systems have been reported, for example in Folkman et al. and Lider et al., in EPO Application 0114589 and J. Clin. Invest. (1989) 83:752:756. In addition, Diferrante, N., in published International Application WO 90/03791, teaches the use of LMWH to inhibit the reproduction of HIV in cultures of C8166 transformed human lymphocytes (ALL). However, none of the prior art experiments that have studied the effects of LMWH on cellular metabolism has considered that the heterogeneity of LMWH may produce antagonistic effects. Furthermore, none has shown or suggested a regulatory effect on cytokine activity based on the use of substantially pure oligosaccharide substances.
Cahalon et al. (International Immunology, vol. 9, p, 1517-1522, 1997; see also Lider et al., Proc. Natl. Acad. Sci. USA, vol 92, p. 5037-5041, 1995) describe the ability of heparin disaccharides to inhibit tumor necrosis factor alpha production by macrophages. These disaccharides are also able to stop the immunologically based inflammation process in rodents. Also, disaccharides derived from heparin or heparan sulfate were shown to block IL-8 and IL-1β secretion by intestinal epithelial cells (Chowers et al., Gastroenterology, vol 120, p. 449-459, 2001) and to modulate chemokine-induced T-cell adhesion to the extracellular matrix (Hershkoviz et al., Immunology, vol 99, p. 87-93, 2000).